HIV vaccines designed to promote cellular immunity by delivering native proteins have not been successful. We showed by ex vivo priming that immunodominant HLA-A2-restricted SL9 epitope of HIV Gag produces an unstable help-independent CTL response, while the subdominant TV9 provokes a CDS'1" response unable to full affinity maturation. The agonist p41 peptide of SL9, identified by probing a large combinatorial peptide library with SL9-specific CD8+ T cells, elicits stable SL9-crossreactive T cells. The agonist TV9p6 of TV9 primes avid and focused tetramer* CTLs. Importantly, they are more effective than TV9-CTLs in lysing virus-infected target cells. We hypothesize that the numerically dominant response to the SL9 epitope actively suppresses potentially more efficacious responses to subdominant peptides. particularly TV9. We propose that the effectiveness of HIV Gag as an immunogen for HLA-A2* carriers can be improved by defining its CTL epitope hierarchy, which will allow manipulation these T cell responses. Here we will evaluate T cell competition for SL9 and TV9 by ex vivo priming of human T cells with DCs transduced to express Gag. Aim 1 will determine whether SL9-T cells are derived from naive or memory/effector precursors from healthy donors, to see whether the overactivated response is an intrinsic property of SL9 or due to mobilization of pre-existing crossreactive memory. Aim 2 will construct four lentiviral vectors in which the SL9- or TV9-regions are modified by point mutations. pL-wtGag will encode wtGag; pL-ASL9 will contain a point mutation to disable binding of SL9 to HLA-A2; pL-p41 will have its SL9 replaced by p41; and the 4th vector will build on the most effective vector defined by Aim 3, in which TV9 is replaced with TV9p6. Aim 3 will determine whether the hierarchy of CTL epitopes determines the ability of the responding cells to suppress viral replication. It will test whether numerically dominant SL9 response actively suppresses more efficacious responses to TV9 and possibly other minor epitopes. T cells will be ranked according to their ability to suppression HIV in vitro. Diversity of the response will be judged by clonotype TCR sequencing. Aim 4 will determine whether the most effective vector will generate protective memory CD8+ when challenged with a surrogate virus in HHD mice. Immunodominance has been studied primarily in mice with model viruses. Our studies will attempt to understand why SL9- and TV9-responses do not appear to play important roles in controlling HIV infections. A new approach, combining studies of the TCR degeneracy for SL9 and TV9 with reverse genetics, will be tested to improve the immunogenicity of HIV Gag. [unreadable] [unreadable] [unreadable]